Nipple angiofibromas with loss of TSC2 are associated with tuberous sclerosis complex

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1 Nipple angiofibromas with loss of TSC2 are associated with tuberous sclerosis complex The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Nathan, Neera, Magdalena E. Tyburczy, Lana Hamieh, Jian Wang, G. Thomas Brown, Chyi-Chia Richard Lee, David J. Kwiatkowski, Joel Moss, and Thomas N. Darling Nipple angiofibromas with loss of TSC2 are associated with tuberous sclerosis complex. The Journal of investigative dermatology 136 (2): doi: /j.jid j.jid Published Version doi: /j.jid Citable link Terms of Use This article was downloaded from Harvard University s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at nrs.harvard.edu/urn-3:hul.instrepos:dash.current.terms-ofuse#laa

2 Nipple angiofibromas with loss of TSC2 are associated with tuberous sclerosis complex Neera Nathan 1,2, Magdalena E. Tyburczy 3, Lana Hamieh 3, Ji-an Wang 1, G. Thomas Brown 4, Chyi-Chia Richard Lee 4, David J. Kwiatkowski 3, Joel Moss 2, and Thomas N. Darling 1 1 Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA 2 Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA 3 Department of Medicine, Brigham and Women s Hospital, Harvard Medical School, Boston, MA, USA HHS Public Access Author manuscript Published in final edited form as: J Invest Dermatol February ; 136(2): doi: /j.jid Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA TO THE EDITOR Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome that leads to hamartoma formation in multiple organs, including the skin ( Curatolo et al., 2008 ). Cutaneous hamartoma formation occurs secondary to loss of function of either the TSC1 or TSC2 gene in fibroblast-like cells and subsequent dysregulation of the mechanistic target of rapamycin complex 1, or mtorc1 ( Li et al., 2008 ; Tyburczy et al., 2014 ). Angiofibromas are among the most well recognized TSC-related skin hamartomas and consist of multiple pink papules on the central face ( Little, 1909 ). Here we describe angiofibromas of the nipple-areolar complex. Between October 2012 and January 2015, 53 TSC patients (50 women, 3 men) participated in studies of lymphangioleiomyomatosis (LAM), a TSC-associated lung disease with female predominance, at the National Institutes of Health in Bethesda, Maryland. Written informed consent was obtained according to IRB-approved protocols 00-H-0051, 95-H-0186 and/or 82-H Eleven TSC patients (10 women, 1 man; median age 41 years [range years]) with papules on the nipple and/or areola were identified (Table), eight of whom had germline mutations in TSC2. Clinical examination revealed 1 to 25, 1 3 millimeter, pink to red domeshaped papules on the nipple and/or areola, affecting one or both breasts (Figure 1a). Per Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: Corresponding author and reprint request: Thomas N. Darling, M.D., Ph.D., Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland, USA 20814, Telephone: , Fax: , ; thomas.darling@usuhs.edu

3 Nathan et al. Page 2 patient recollection, the median age of onset was 33 years (n=7 women; range years). One patient reported painless bleeding from her nipple papules during breastfeeding while the rest remained asymptomatic. Histopathological examination of ten biopsied nipple or areolar papules from seven patients revealed increased number of dilated capillaries in the papillary dermis and increased number of stellate and spindle-shaped fibroblasts interspersed in the dermal collagen, consistent with the diagnosis of an angiofibroma (Figure 1b). Increased numbers of factor XIIIa positive spindle to stellate shaped cells were observed in the dermis of the nipple-areolar complex papules (Figure 1c), as characteristically observed in TSC-related facial angiofibromas ( Li et al., 2005 ). To detect activation of mtorc1 in nipple-areolar complex papules, immunohistochemical staining against phosphorylated ribosomal protein S6 (ps6) (Ser235/236) was performed on ten samples, revealing avid positive dermal fibroblast-like cells as seen in other TSC-related skin hamartomas (Figure 1d) ( Li et al., 2008 ). Western blot analysis of fibroblast-like cells grown from two out of four nipple angiofibromas demonstrated loss or near complete loss of TSC2. All four showed increased ps6 under serum starvation, compared to dermal fibroblasts from normal-appearing skin (Supplementary Figure 1), consistent with prior molecular analysis of facial angiofibromas ( Tyburczy et al., 2014 ). Mutational analysis of DNA extracted from papule-derived fibroblast-like cells or whole tissue biopsies was performed as described previously using targeted next generation sequencing (NGS) of TSC1 and TSC2, with validation by Sanger sequencing or SNaPshot analysis ( Tyburczy et al., 2014 ). Seven angiofibromas from five patients were analyzed and biallelic mutations in TSC2 were identified in four samples (Table, Supplementary Figure 2). A nonsense mutation in TSC2 (p.arg1459*) was present in one nipple angiofibroma fibroblast culture at 99% allele frequency, consistent with both a germline p.arg1459* mutation and second hit loss of the wild-type allele (Table, P27, Supplementary Figure 2a). Three samples (2 whole tissue, 1 cultured cells; Table, P36 and P37) had second hit point mutations in TSC2 that were seen at low allele frequency (3% to 5%), albeit undetectable in patient controls, consistent with the low prevalence of neoplastic fibroblast cells in these biopsies. All three of the low frequency second hit mutations seen in these samples were validated by an independent method (Supplementary Figure 2e j). The remaining samples in which neither second hit point mutations nor loss of heterozygosity (LOH) was seen in TSC2 may be due to the occurrence of LOH, which is very difficult to detect in a sample in which the tumor cells are at low frequency. This microscopic and molecular evidence of mtorc1 activation with inactivating mutations in TSC2 provides strong support for the concept that these lesions arise through two hit inactivation of TSC1/ TSC2, as we have shown previously for facial angiofibromas ( Tyburczy et al., 2014 ). In our study, the frequency of nipple-areolar complex angiofibromas in adult women was 20%. Median age of onset was 33 years (range 16 38), nearly three decades after onset of facial angiofibromas in the same female patients (median age 4 years; range 3 13). This indicates that nipple-areolar complex angiofibromas are an adult manifestation of TSC, occurring later than any cutaneous feature currently described. In addition, the presence of

4 Nathan et al. Page 3 nipple and areolar angiofibromas in a man from our cohort suggests that unlike LAM, these hamartomas are not limited to women. The differential diagnosis of nipple-areolar complex papules in a TSC patient should include several benign tumors, such as nipple adenomas/ florid papillomatosis, leiomyomas, fibromas, and folliculosebaceous cystic hamartomas ( Badr and Lakshmiah, 2009 ; Spyropoulou et al., 2014 ). For patients without known TSC, consideration should also be given to nipple papules and nodules related to other tumor syndromes, including myxomas and neurofibromas ( Bongiorno et al., 2010 ; Carney et al., 1986 ). Biopsy and pathologic examination are valuable to confirm the diagnosis of angiofibroma, both for diagnostic purposes and for reassurance in regards to natural history. Treatment may not be necessary; unlike facial angiofibromas, angiofibromas of the nipple and areola do not bleed spontaneously. Nipple-areolar complex angiofibromas were less common than facial angiofibromas in our cohort, affecting 20% versus 100% of female patients, respectively. Less frequent occurrence of angiofibromas on the nipple or areola may be explained by protection from sun exposure as our group previously demonstrated that signature second hit UV mutations promote facial angiofibroma formation ( Tyburczy et al., 2014 ). We propose that repetitive trauma from breastfeeding may stimulate hamartoma development in female patients, just as shoe wear compression may stimulate ungual fibroma pathogenesis ( Aldrich et al., 2010 ). One patient reported onset of a nipple papule in the weeks following initiation of breastfeeding and two additional patients reported onset of papules after discontinuing breastfeeding. Thus, both somatic mutations and trauma may drive TSC-related skin tumor pathogenesis. In summary, we report nipple-areolar complex angiofibromas, a cutaneous manifestation of TSC. Similar to facial angiofibromas, they occur following second hit loss of the wild type allele of TSC2, but manifest much later in life. They have a benign clinical course, with symptomatic manifestations only associated with local trauma. When verified by histologic analysis, they may provide additional information in favor of TSC diagnosis. Supplementary Material Acknowledgments Abbreviations Refer to Web version on PubMed Central for supplementary material. This work was supported by NIH R01AR and 1P01CA120964, the Sulzberger Dermatological Research and Education Endowment, the Doris Duke Charitable Foundation # , European Commission , and the Intramural Research Program of the National Institutes of Health, National Heart, Lung, and Blood Institute. TSC LAM ps6 tuberous sclerosis complex lymphangioleiomyomatosis phosphorylated ribosomal protein S6 mtorc1 mechanistic target of rapamycin complex 1

5 Nathan et al. Page 4 References NGS LOH next generation sequencing loss of heterozygosity Aldrich CS, Hong CH, Groves L, et al. Acral lesions in tuberous sclerosis complex: insights into pathogenesis. J Amer Acad Dermatol. 2010; 63: [PubMed: ] Badr A, Lakshmiah GR. Folliculosebaceous cystic hamartoma of the nipple: a case report. J Cutan Pathol. 2009; 36: [PubMed: ] Bongiorno MR, Doukaki S, Arico M. Neurofibromatosis of the nipple-areolar area: a case series. J Med Case Reps. 2010; 4:22. Carney JA, Headington JT, Su WP. Cutaneous myxomas. A major component of the complex of myxomas, spotty pigmentation, and endocrine overactivity. Arch Dermatol. 1986; 122: [PubMed: ] Curatolo P, Bombardieri R, Jozwiak S. Tuberous sclerosis. Lancet. 2008; 372: [PubMed: ] Li S, Takeuchi F, Wang JA, et al. Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas. Proc Natl Acad Sci U S A. 2008; 105: [PubMed: ] Li S, Takeuchi F, Wang JA, et al. MCP-1 overexpressed in tuberous sclerosis lesions acts as a paracrine factor for tumor development. J Exp Med. 2005; 202: [PubMed: ] Little EG. Adenoma Sebaceum in a Girl aged 20. Proc R Soc Med. 1909; 2: [PubMed: ] Spyropoulou GA, Pavlidis L, Trakatelli M, et al. Rare benign tumours of the nipple. J Eur Acad Dermatol Venereol. 2014; 29:7 13. [PubMed: ] Tyburczy ME, Wang JA, Li S, et al. Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex. Hum Mol Gen. 2014; 23: [PubMed: ]

6 Nathan et al. Page 5 Figure 1. Gross and microscopic features of nipple-areolar complex angiofibromas (a) Pink papules on the nipple and areola. (b) Hematoxylin and eosin stain shows increased number of dilated vessels and fibroblasts consistent with the diagnosis of an angiofibroma. (c) Factor XIIIa staining demonstrates positive cells in the dermis as seen in facial angiofibromas. (d) Immunoreactivity to phosphorylated ribosomal protein S6 (ps6) in scattered dermal fibroblast-like cells suggests hyperactivation of the mechanistic target of rapamycin.

7 Nathan et al. Page 6 Table Clinical, microscopic and mutational analysis of nipple-areolar complex angiofibromas. Patient Sex TSC cutaneous major features History of breastfeeding Lesions on nipple (biopsied) Lesions on areola (biopsied) Histopathology Tissue for mutational analysis TSC2 mutation (af) TSC2 protein change P25 F AF, HM no 1 13 (1) angiofibroma P27 F AF, FCP yes 3 (1) 3 angiofibroma, associated hidrocystoma cultured fibroblasts c.4375c>t (.99) p.(arg1459*) P28 F AF, SP, FCP yes 3 (1) 0 angiofibroma, prominent vascular component cultured fibroblasts c.4868_4875delcc CTGATG (.54) Out-of-frame deletion P34 F AF, HM, UF no 1 0 P35 F AF, HM, UF, FCP no 1 0 P36 F AF, HM, UF yes 5 (1) 5 (1) angiofibroma cultured fibroblasts c g>a (.5) c.4858c>t (.05) p.(his1620tyr) P37 F AF, HM, UF yes 11 (2) 14 (1) angiofibroma whole tissue T1 c g>a (.5) c.138+1g>a (.05) whole tissue T2 c g>a (.5) c.3574c>t (.03) p.(gln1192*) whole tissue T3 c g>a (.5) P43 F AF, UF no 3 (1) 0 angiofibroma cultured fibroblasts c.3999c>a (.5) p.(tyr1333*) P46 F AF, HM, UF no 1 3 P47 F AF, HM, UF yes 1 (1) 0 angiofibroma P48 M AF, HM, UF 11 0 Abbreviations: AF, angiofibromas; af, allelic frequency; F, female; FCP, fibrous cephalic plaque; HM, hypomelanotic macules; L, left; M, male; R, right; SP, shagreen patch; TSC, tuberous sclerosis complex; UF, ungual fibromas. Bold denotes germline mutation when documented in blood or control skin.